CN1330770C - Starch process - Google Patents

Starch process Download PDF

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CN1330770C
CN1330770C CNB038039869A CN03803986A CN1330770C CN 1330770 C CN1330770 C CN 1330770C CN B038039869 A CNB038039869 A CN B038039869A CN 03803986 A CN03803986 A CN 03803986A CN 1330770 C CN1330770 C CN 1330770C
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ser
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CN1633503A (en
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巴里·E·诺曼
安德斯·维克索-尼尔森
汉斯·S·奥尔森
斯文·佩德森
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Novozymes AS
Novo Nordisk AS
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Priority to PCT/DK2003/000084 priority patent/WO2003068976A2/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/20Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/14Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/18Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/22Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

Abstract

The present invention relates to a process for enzymatic hydrolysis of granular starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch.

Description

Starch process
Invention field
The present invention relates in the single stage method that granular starch hydrolyzing is become the Zulkovsky starch hydrolysate, this method is carried out under the temperature of the initial gelation temperature that is lower than described granular starch.
Background of invention
Described many with starch change into starch hydrolysate, such as maltose, glucose or extraordinary syrupy method, these hydrolysates both can be used as sweetener, also can be as such as other sugared precursor of this class of fructose.Glucose fermentation can also be become alcohol or other tunning.
The high-molecular weight polymer that starch is made up of glucose unit.It is made up of about 80% amylopectin and 20% amylose starch usually.Amylopectin is the side chain polysaccharide, α-1 wherein, and the straight chain of 4D-glucosyl residue connects by α-1,6 glycosidic link.
The amylose starch straight-chain polysaccharide that the D-Glucopyranose unit that connects by α-1,4 glycosidic link constitutes of serving as reasons each other.Starch is being changed in the situation of Zulkovsky starch hydrolysate the starch depolymerization.Depolymerization commonly used by gelling step and two successive processing steps, be that liquefaction process and Mashing process are formed.
Granular starch is made up of water-fast microscopic particles at room temperature.When with aqueous starch when heating slurry, particle swelling and finally breaking, thus starch molecule is scattered in the solution.In this " gelling " technological process, viscosity significantly increases.When solids concn is 30-40% in the typical industrial method, must make starch thinning or " liquefaction " so that can operate on it.Mainly viscosity is reduced at present by enzymatic degradation.In the liquefaction step process, α-Dian Fenmei becomes less side chain and straight chain unit's (Star Dri 5) with the long-chain starch degradation.Liquefaction step is generally under about 105-110 ℃ about 5-10 minute, under about 95 ℃ about 1-2 hour subsequently.Make temperature reduce to 60 ℃ then, add glucoamylase or beta-amylase and optional debranching factor, such as isoamylase or Starch debranching enzyme and make the about 24-72 of saccharification step hour.
Obviously can see that from above-mentioned discussion conventional starch conversion method extremely consumes energy because of the demand to temperature in various steps.Whole technology needs thus to select used in the method enzyme so that can be carried out needn't making under the starch agglomerative condition.These class methods are themes of US4591560, US4727026 and US4009074 and EP0171218 patent.
The present invention relates to granular starch is changed into the single stage method of Zulkovsky starch hydrolysate under the temperature of the initial gelation temperature that is lower than this starch.
Summary of the invention
The present invention provides the single stage method that is used to produce the Zulkovsky starch hydrolysate in aspect first, this method comprises the step that makes the aqueous particulate starch slurry carry out effect in the following enzymic activity under the temperature of the initial gelation temperature that is lower than described granular starch: first kind of enzyme, it be the 13rd family's glycosides lytic enzyme the member, to have α-1.4-glycoside hydrolysis active and comprise the 20th family's carbohydrate binding constituents (Carbohydrate-Binding Module Family 20); With second kind of enzyme, it is fungal alpha-amylase (EC 3.2.1.1), beta-amylase (E.C.3.2.1.2) or glucoamylase (E.C.3.2.1.3).
The present invention is in second production method that provides in aspect this based on the syrup (HFSS) of high fructose starch, and this method comprises that the method for using first aspect of the present invention produces the Zulkovsky starch hydrolysate and further comprise this Zulkovsky starch hydrolysate is changed into step based on the syrup (HFSS) of high fructose starch.
The present invention provides fuel alcohol or drinkable production method of alcohol the 3rd in aspect this, this method comprises the method production Zulkovsky starch hydrolysate that uses first aspect of the present invention and further comprises the step that this Zulkovsky starch hydrolysate is fermented into alcohol that wherein the hydrolysing step of fermentation step and granular starch carries out at the same time or separately/successively.
Detailed Description Of The Invention
Definition
Term " granular starch " should be understood the not starch (raw uncooked starch) of heat system of making a living, promptly not carry out agglomerative starch.Starch forms in plant as small water-fast particle.These particles are protected in starch being lower than under the temperature of initial gelation temperature.When putting into cold water, these particles can absorb small amount of liquid.Under up to 50 ℃-70 ℃, swelling is a reversible, and reversible degree depends on specific starch.Under higher temperature, be called the agglomerative irreversible swelling and begin to take place.
Term " initial gelation temperature " should be interpreted as that starch begins the agglomerative minimum temperature.Starch begins gelling between 60 ℃-70 ℃, definite temperature depends on concrete starch.Initial gelation temperature depends on the source of institute's starch producing.The initial gelation temperature of wheat is about 52 ℃, and the initial gelation temperature of potato is about 56 ℃, and the initial gelation temperature of corn is about 62 ℃.Yet starch primary quality changes with growth conditions different and tackles every batch of initial gelation temperature of starch test thus with the specified plant kind.
" Zulkovsky starch hydrolysate " should be interpreted as the soluble product of the inventive method and can comprise monose, disaccharides and oligosaccharides class, such as glucose, maltose, Star Dri 5, cyclodextrin and mixture arbitrarily thereof.Preferably general at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% the dried solid of granular starch changes into the Zulkovsky starch hydrolysate.
Term " special syrup " be term well-known in the art and by DE and carbohydrate spectral characterization (referring to the article of p.50+ going up in the textbook " Molecular Structure and Function of Food Carbohydrate " " new special glucose syrup ", G.G.Birch and L.F.Green edit, AppliedScience Publishers LTD., London).In general, special syrup has the DE of 35-45 scope.
In the context of the present invention, " the 13rd family's glycosides lytic enzyme " is defined as comprises having (beta/alpha) 8Or the lytic enzyme family of TIM bucket (barrel) structure and the catalyst structure domain that starch and related substrates worked by α-reservation reaction mechanism (Koshland, 1953, Biol.Rev.Camp.Philos.Soc 28,416-436).
In the context of the present invention, the enzyme that will have " α-1.4-glycoside hydrolysis activity " is defined as the Takata (Takata etc. that comprise by definition, 1992, J.Biol.Chem.267,18447-18452) and Koshland (Koshland, 1953, Biol.Rev.Camp.Philos.Soc 28,416-436) catalysis α-1.4-glycosidic link hydrolysis and/or synthetic enzyme family.
In the context of the present invention, " the 20th family's carbohydrate binding constituents " or CBM-20 composition are defined as about 100 the amino acid whose sequences that have at least 45% homology with (1997) carbohydrate binding constituents (CBM) of disclosed polypeptide in Biotechnol.Lett.19:1027-1031 Fig. 1 such as Joergensen.CBM comprises 102 the most last amino acid of this polypeptide, promptly from the subsequence of the 582nd amino acids-the 683rd amino acids.
(a) belonging to the 13rd member of glycosides lytic enzyme family of family, (b), to have α-1.4-glycoside hydrolysis active and (c) comprise the 20th family's carbohydrate binding constituents, and comprise the enzyme that is categorized as EC 2.4.1.19, be cyclodextrin glucanotrasferase enzyme and EC 3.2.1.1 33, be the selection member in Fructus Hordei Germinatus sugar α-Dian Fenmei and 3.2.1.1 α-Dian Fenmei and the 3.2.1.60 maltotetrose formation amylase for the enzyme of special concern of the present invention.
By in the starch insulating process according to Wind, R.D. etc. 1995 determine " hydrolytic activity " of CGTases and Fructus Hordei Germinatus sugar α-Dian Fenmei in the increase of the described mensuration reducing power of Appl.Environ.Microbiol.61:1257-1265 (reducing power).The dinitrosalicylic acid method of using Bernfield (Bernfield, P.1955.Amylases alpha and beta.Methods Enzymol.1:149-158) is through suitably revising the concentration of measuring reducing sugar.Under 60 ℃ the enzyme of dilution is being incubated the appropriate time time limit with 1% (wt/v) Zulkovsky starch (maybe can select Zulkovsky starch from Merck from the Paselli SA2 starch of Dutch Avebe) in 10mM Trisodium Citrate (pH5.9) damping fluid.The hydrolytic activity of a unit is defined as the enzyme amount that under standard conditions per minute produces 1 micromole's maltose.
The polypeptide " homology " that relates in this manual is interpreted as that first kind of sequence of expression is from the identity degree between two kinds of sequences of second kind of sequence deutero-.Can suitably measure homology by computer program as known in the art, such as the GAP that provides in the GCG routine package (Wisconsin Package procedure manual, Version 8, August, 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48,443-453.Use following to peptide sequence setting relatively: GAP generation compensation (penalty) 3.0 and GAP expansion are compensated for as 0.1.
Cyclodextrin glucanotrasferase enzyme (CGTases)
As the concrete enzyme of first kind of enzyme in the inventive method can be for cyclomaltodextrin glucanotransferase (CGTASE) (E.C.2.4.1.19), be also referred to as cyclodextrin glucanotrasferase enzyme or Maltose 4-glucosyltransferase, be called CGTase hereinafter, it changes the glycosyl reaction by intramolecularly starch is catalytically converted into the ring Star Dri 5 with similar substrate, forms the ring Star Dri 5 of all size thus.Most of CGTases had not only had the glycosyl of commentaries on classics activity, but also had had the activity of degraded starch.The CGTases that is paid close attention to preferably derives from microorganism and most preferably derives from bacterium.The CGTases of special concern comprises: the CGTases that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology with sequence shown in the 1-679 amino acids of SEQ ID NO:2 among the WO02/06508; With 1997 CGTases that disclosed polypeptid acid sequence has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% homology in Biotechnol.Lett.19:1027-1031 Fig. 1 such as Joergensen; And the CGTases described in US5278059 and the US5545587.Preferably the hydrolytic activity that has of the CGTase that uses as first kind of enzyme of described method be at least 3.5, preferably be at least 4,4.5,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 or most preferably be at least 23 micromoles/minute/mg.The DS CGTases that can add 0.01-100.0 NU/g DS, preferred 0.2-50.0NU/g DS, preferred 10.0-20.0 NU/g consumption.
Fructus Hordei Germinatus sugar α-Dian Fenmei
As the another kind of concrete enzyme of first kind of enzyme of the inventive method maltogenic alpha-amylase enzyme (E.C.3.2.1.133) of making a living.Fructus Hordei Germinatus sugar α-Dian Fenmei (dextran 1,4-α-maltose lytic enzyme) can be hydrolyzed into amylose starch and amylopectin the maltose of α-configuration.In addition, Fructus Hordei Germinatus sugar α-Dian Fenmei can hydrolysis trisaccharide maltose and cyclodextrin.The Fructus Hordei Germinatus of special concern sugar α-Dian Fenmei can derive from the kind of bacillus, preferably derive from bacstearothermophilus, most preferably derive from such as the bacstearothermophilus C599 that is described among the EP120.693.This specific Fructus Hordei Germinatus sugar α-Dian Fenmei has the aminoacid sequence (being the SEQ ID NO:5 of present specification sequence table) shown in the 1-686 amino acids of SEQ ID NO:1 among the US6162628.The 1-686 amino acids of SEQ ID NO:1 has at least 70% identity, preferred at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or particularly at least 99% identity among aminoacid sequence that preferred Fructus Hordei Germinatus sugar α-Dian Fenmei contains and the US6162628.The variant of most preferred Fructus Hordei Germinatus sugar α-Dian Fenmei comprises disclosed variant among the WO99/43794.
Contain and have 714 hydrolytic activity just like the Fructus Hordei Germinatus of the aminoacid sequence shown in the 1-686 amino acids of SEQ ID NO:1 among US6162628 sugar α-Dian Fenmei.The Fructus Hordei Germinatus sugar α-Dian Fenmei that is preferably used as first kind of enzyme of the inventive method have at least 3.5, preferably at least 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,100,200,300,400,500,600 or most preferably at least 700 micromoles/minute/hydrolytic activity of mg.
The Fructus Hordei Germinatus sugar α-Dian Fenmei that can add 0.01-40.0 MANU/g DS, preferred 0.02-10 MANU/g DS, preferred 0.05-5.0 MANU/g consumption.
Fungal alpha-amylase
Concrete enzyme as second kind of enzyme of the inventive method is fungal alpha-amylase (EC 3.2.1.1), adds Mill sample (Fungamyl-like) α-Dian Fenmei such as sweet smell.In this manual, term " sweet smell adds Mill sample α-Dian Fenmei " refers to aminoacid sequence shown in the SEQ ID No.10 that shows with WO96/23874 and has high homology, promptly has a α-Dian Fenmei of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% above homology with aminoacid sequence shown in the SEQ ID No.10 of WO96/23874.The fungal alpha-amylase that can add 0.001-1.0 AFAU/g DS, preferred 0.002-0.5 AFAU/g DS, preferred 0.02-0.1 AFAU/g DS consumption.
Beta-amylase
Another kind of concrete enzyme as second kind of enzyme in the inventive method is beta-amylase (E.C 3.2.1.2).Beta-amylase is the title that generally malt amylase that plays circumscribed effect is given, 1 on its catalysis amylose starch, amylopectin and the relevant glucose polymer, the hydrolysis of 4-α-glycosidic link.
From each kind of plant and microorganism, separated beta-amylase (W.M.Fogarty and C.T.Kelly, Progress in Industrial Microbiology, vol.15, pp.112-115,1979).These beta-amylases are characterised in that the optimum temps of 40 ℃ of-65 ℃ of scopes of tool and the best pH of 4.5-7.0 scope.The beta-amylase of paying close attention to comprises from barley Spezyme  BBA 1500, Spezyme  DBA and Optimalt TMThe beta-amylase of ME, from the Optimalt of Genencor int TMBBA and from the Novozym of Novozymes A/S TMWBA.
Glucoamylase
Also can be as the another kind of concrete enzyme of second kind of enzyme in the inventive method for deriving from microorganism or deriving from the glucoamylase (E.C.3.2.1.3) of plant.Preferably derive from the glucoamylase of fungi or bacterium, they are selected from Aspergillus glucoamylase, particularly black aspergillus G1 or G2 glucoamylase (Boel etc. (1984), EMBO is (5) J.3, p.1097-1102) or it is such as the variant, Aspergillus awamori glucoamylase (WO84/02921), the aspergillus oryzae (Agric.Biol.Chem. (1991) that are disclosed among WO92/00381 and the WO00/04136,55 (4), p.941-949) or the group formed of its variant or fragment.
The Aspergillus glucoamylase variant of other concern comprise the variant that improves thermostability: G137A and G139A (Chen etc. (1996), Prof.Engng.9,499-505); D257E and D293E/Q (Chen etc. (1995), Prot.Engng.8,575-582); N182 (Chen etc. (1994), Biochem.J.301,275-281); Disulfide linkage A246C (Fierobe etc. (1996), Biochemistry, 35,8698-8704; With the Pro residue that imports on A435 and the S436 position (Li etc. (1997), Protein Engng.10,1199-1204.In addition, Clark Ford is at the ENZYMEENGINEERING 14 on October 17th, 1997, Beijing/China October, and 12-17,97, the summary collected works provide in p.0-61 and have been paper.Proposed in this summary to improving thermostability sudden change on G137A, N20C/A27C and the S30P position in the Aspergillus awamori glucoamylase.The glucoamylase of other concern comprises the Talaromyces glucoamylase, particularly derive from Talaromycesemersonii (WO99/28448), Talaromyces leycettanus (US patent no.Re.32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no.4,587,215) glucoamylase.The bacterium glucoamylase of paying close attention to comprises the glucoamylase from Clostridium, particularly C.thermoamylolyticum (EP135,138) and the hot anaerobic bacillus(cillus anaerobicus) of hot sulfurization hydrogen (WO86/01831).Preferred glucoamylase comprises the glucoamylase that derives from aspergillus niger, such as the glucoamylase that has 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and even 90% identity with aminoacid sequence (being the SEQ ID NO:6 of present specification sequence table) shown in the SEQ ID NO:2 of WO00/04136.That pay close attention in addition is commodity AMG 200L, AMG 300L, SAN TMSUPER and AMG TME (from Novozymes), OPTIDEX TM300 (from Genencor Int.), AMIGASE TMAnd AMIGASE TMPLUS (from DSM), G-ZYME TMG900 (from Enzyme Bio-Systems), G-ZYME TMG990 ZR (black aspergillus glucoamylase and low protease content).
Can add 0.02-2.0 AGU/g DS, preferred 0.1-1.0 AGU/gDS, such as the glucoamylase of 0.2 AGU/gDS consumption.
Other enzyme
Can also under the situation that has the third enzyme to exist, implement method of the present invention.The third concrete enzyme can be the α-Dian Fenmei (so-called " saturating A Mier sample (Termamyl-like) α-Dian Fenmei ") of bacillus.Well-known A Mier sample α-Dian Fenmei comprises the α-Dian Fenmei that derives from Bacillus licheniformis (being purchased as saturating A Mier), bacillus amyloliquefaciens and bacstearothermophilus bacterial strain α-Dian Fenmei.Other saturating A Mier sample α-Dian Fenmei comprise the α-Dian Fenmei that derives from bacillus kind NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375 bacterial strains of whole specific descriptions in WO95/26397 and by Tsukamoto etc. at Biochemical and Biophysical ResearchCommunications, 151 (1988), the α-Dian Fenmei of describing among the pp.25-31.In the context of the present invention, saturating A Mier sample α-Dian Fenmei is that page 3 the 18th walks to the α-Dian Fenmei that the 6th page of the 27th row upward defines among the WO99/19467.Variant and the hybrid paid close attention to are described among WO96/23874, WO97/41213 and the WO99/19467.Special concern contains sudden change I181 *+ G182 *The reorganization bacstearothermophilus alpha-amylase variants of+N193F.The bacillus α-Dian Fenmei that can add the well-known significant quantity of those skilled in the art.
The third concrete enzyme of another kind in the described method can be debranching factor, such as isoamylase (E.C.3.2.1.68) or Starch debranching enzyme (E.C.3.2.1.41).α on isoamylase hydrolysis amylopectin and the β-limit dextrin-1,6-D-glucosides branching key and can be able to not and aspect the limited action of α-limit dextrin, be different from Starch debranching enzyme with Aureobasidium pullulans glycan generation chemical reaction.The debranching factor that can add the well-known significant quantity of those skilled in the art.
Embodiment of the present invention
The starch slurry that is used to implement the inventive method can contain the dried solid particle starch of the dried solid particle starch of 20-55%, preferred 25-40%, the more preferably dried solid particle starch of 30-35%.
After the method for implementing aspect the present invention the-individual, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferred 99% dried solid particle starch is converted to the Zulkovsky starch hydrolysate.
The method of the present invention first and second aspect is carried out being lower than under the temperature of initial gelation temperature.The preferred temperature of implementing this method is at least 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ or preferably at least 60 ℃.
The pH that implements first aspect of the present invention can be in 3.0-7.0, preferred 3.5-6.0 or the more preferably scope of 4.0-5.0.
The definite composition of the product Zulkovsky starch hydrolysate in the method for first aspect of the present invention depends on the combination of used enzyme and the type of the granular starch of being processed.Preferred solubility hydrolysate is the maltose with at least 85%, 90%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5,99.0% or 99.5% purity.Even more preferably the Zulkovsky starch hydrolysate be glucose and most preferably starch hydrolysate have at least 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5,99.0% or 99.5% DX (per-cent of glucose in the dried solid of dissolved).But, focus attentions equally on be that product Zulkovsky starch hydrolysate in the inventive method is the method for special syrup (specialty syrup), the special syrup that is used for ice-creams, cake, candy, fruit can of described special syrup such as the mixture that contains glucose, maltose, DP3 and DPn.
The granular starch of processing in the inventive method especially can be available from stem tuber, root, stem, beans, cereal or full cereal.More particularly, granular starch can be available from corn, corn cob (cobs), wheat, barley, rye, milo (milo), sago (sago), cassava (cassava), tapioca (flour) (tapioca), jowar, rice, pea, beans, banana or potato.Special concern wax class and non-wax class corn and barley.The granular starch of being processed can have for highly refined starch quality, preferred purity be higher than 90%, 95%, 97% 99.5% or its can be for containing the thicker starch of the material that comprises the full cereal of ground, the full cereal of described ground comprises non-starch part, such as plumule nubbin and fiber.Grinding such as the so thick material of full cereal is in order to make structure opening and further to process.The preferred two kinds of polishings of the present invention: wet-milling and dry grinding.In the dry grinding method, grind complete grain and use.Wet milling process can make plumule and meal (starch granules and protein) obtain good separation and this method can be used for the situation that any production syrup except that a few cases uses starch hydrolysate.Dry grinding and wet-milling are well-known and focus attentions equally on methods of the present invention in the starch manufacture field.The method of first aspect of the present invention can be carried out in ultrafiltration system, and wherein phegma has remained on enzyme.Give birth in the circulation that starch (raw starch) and water exists and be the Zulkovsky starch hydrolysate wherein through thing.The method that focus attentions equally on carries out in having the continuous film reactor of ultra-filtration membrane and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.Also focus on the method for carrying out in the continuous film reactor that has microfiltration membrane and wherein phegma to remain on enzyme, give birth in the circulation that starch and water exists and wherein see through thing be the Zulkovsky starch hydrolysate.
In the method aspect second of the present invention, the Zulkovsky starch hydrolysate in first aspect method of the present invention is changed into syrup (HFSS) based on high fructose starch, such as high fructose corn syrup (HFCS).Preferably use glucose isomerase and more preferably carry out this conversion by the fixed glucose isomerase of supporting on the solid support.The isomerase of paying close attention to comprises the commodity Sweetzyme from Novozymes A/S TMIT, from the G-zyme of Rhodia TMIMGI and G-zyme TMG993, Ketomax TMAnd G-zyme TMG993, from the G-zyme of Genemcor Int TMG993 liquid and GenSweet TM
In the present invention the 3rd method aspect this, the Zulkovsky starch hydrolysate in first aspect method of the present invention is used to produce fuel alcohol or drinkable alcohol.In the method aspect the 3rd, fermentation can be carried out at the same time or separately/successively with the hydrolysis of granular starch slurry.When fermentation and hydrolysis were carried out simultaneously, temperature was preferably at 30 ℃-35 ℃ and more preferably at 31 ℃-34 ℃.The method of third aspect of the present invention can be carried out in ultrafiltration system, in the circulation that wherein phegma remains on enzyme, gives birth to starch, yeast, yeast nutrition thing and water exist and wherein to see through thing be the liquid that contains alcohol.In method that focus attentions equally on carries out in having the continuous film reactor of ultra-filtration membrane and the circulation that wherein phegma remains on enzyme, gives birth to starch, yeast, yeast nutrition thing and water exist and wherein to see through thing be the liquid that contains alcohol.
Material and method
Alpha-amylase activity (KNU)
Use yam starch to measure amylolytic activity as substrate.This method is mixed with iodine solution to the decomposition of the yam starch of modification and at this reaction back slurry starch/enzyme solution sample based on enzyme.Begin to form black-and-blue, but in amylolytic process, blueness shoal and progressively become pale red brown, with itself and tinted shade standard substance relatively.
A Kilo Novo α-Dian Fenmei unit (KNU) is defined as under standard conditions (promptly 37 ℃+/-0.05; 0.0003 M Ca 2+Under pH5.6) make the enzyme dosage of 5.26g Zulkovsky starch dry-matter dextrinization.
The file AF 9/6 that more specifically describes this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.
CGTase activity (KNU)
Measure the CGTase alpha-amylase activity by using Phadebas  sheet as substrate.Phadebas sheet (Phadebas  amylase test is provided by Pharmacia Diagnostic) contains and bovine serum albumin(BSA) and the crosslinked insoluble blue starch polymer of buffering material blended.
For each single mensuration, a slice is suspended in contains 5ml 50mM Britton-Robinson damping fluid (50mM acetate, 50mM phosphoric acid, 50mM boric acid, 0.1mMCaCl 2, with NaOH with pH regulator to significant value) pipe in.Carry out in the water-bath of this test under the concern temperature.50mM Britton-Robinson damping fluid with x ml dilutes the α-Dian Fenmei of being tested.This α-Dian Fenmei solution of 1ml is joined in the 5ml 50mM Britton-Robinson damping fluid.Use α-Dian Fenmeishuixie starch, obtain the soluble blue color part.The optical density of the gained blue solution at the 620nm place by spectrophotometry is the function of alpha-amylase activity.
Importantly the 620nm place optical density of measuring in 10 or 15 minutes (test duration) of insulation back is 0.2-2.0 optical density scope at the 620nm place.In this optical density scope, linear between activity and the optical density (Lambert-Beer law).Necessary thus regulatory enzyme diluent is to be fit to this standard.Under one group of actual conditions (temperature, pH, reaction times, buffer conditions), the specified α-Dian Fenmei of 1mg can a certain amount of substrate of hydrolysis and generation blueness.Measure colour intensity at the 620nm place.The optical density of measuring is directly proportional under a specified set condition with the activity specific (the pure α-Dian Fenmei albumen of activity/mg) of described α-Dian Fenmei.
The file EAL-SM-0351 that more specifically describes this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.
Fructus Hordei Germinatus sugar alpha-amylase activity (MANU)
A malt amylase Novo unit (MANU) is defined as the enzyme amount of per minute cracking 1 micromole's trisaccharide maltose under standard conditions.Standard conditions are 10mg/ml trisaccharide maltose, 37 ℃, pH5.0 and 30 minute reaction times.(GlucDH Merck) will change into glucono-lactone with the glucose that forms, and pass through spectrophotometry at the 340nm place with Hexose phosphate dehydrogenase under the condition that forms NADH.The file (EAL-SM-0203.01) of more specifically describing this analytical procedure is available from Novozymes A/S, and Denmark is included in this document herein as a reference.
Glucoamylase activity (AGU)
Novo glucose starch unit of enzyme (AGU) is defined as the enzyme amount of per minute cracking 1 micromole's maltose under 37 ℃ and pH4.3.
With using from Boehringer Mannheim, the method that revise (AEL-SM-0131 obtains from Novozymes as required) of 124036 glucose GOD-Perid test kit back is AGU/ml with determination of activity.Standard substance: AMG-standard substance, lot number 7-1195,195 AGU/ml.Under 37 ℃ with the 375microL substrate (1% maltose in the 50mM sodium acetate, pH4.3) insulation 5 minutes.The 25microL enzyme that adding is diluted with sodium acetate.Make reaction terminating by adding 100microL 0.25MNaOH after 10 minutes.Change 20microL over to 96 hole microtiter plates and add 200microLGOD-Perid solution (124036, Boehringer Mannheim).After at room temperature 30 minutes, in 650nm place mensuration optical density and according to the activity of AMG-standard substance calculating by AGU/ml.The file (AEL-SM-0131) of more specifically describing this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.
Fungal alpha-amylase activity (FAU)
Measure alpha-amylase activity with FAU (fungal alpha-amylase activity unit).The FAU of a unit (1) is for per hour decomposing 5260mg solid starch (Amylum solubile, enzyme amount Merck) under standard conditions (promptly under 37 ℃ and the pH4.7).The file AF9.1/3 that more specifically describes this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.
Acid alpha-amylase activity (AFAU)
Measure the acid alpha-amylase activity with AFAU (acid fungal alpha-amylase activity unit), their contrast enzyme standard substance are measured.
Used standard substance are AMG 300L (from Novozymes A/S, glucoamylase wild-type black aspergillus G1 also is disclosed in (1984) such as Boel, and EMBO is (5) J.3, p.1097-1102WO92/00381 in).Neutral α-Dian Fenmei in this AMG drops to below 0.05 FAU/mL from about 1 FAU/mL after at room temperature storing for 3 weeks.
Measure acid alpha-amylase activity in this AMG standard substance according to following description.In this method, 1 AFAU is defined as the enzyme amount that under standard conditions, per hour makes the dried solid degraded of 5.26mg starch.
Iodine and starch and do not form blue mixture with its degraded product.Colour intensity is directly proportional with starch concentration thus.Use anti-phase colorimetry amylase activity to be determined as the decline of starch concentration under the concrete analysis condition.
α-Dian Fenmei
Starch+iodine → dextrin+oligosaccharides class
40℃,pH2.5
Blueness/purple t=23 decolours second
Standard conditions/reaction conditions: (per minute)
Substrate starch, about 0.17g/L
The damping fluid Citrate trianion, about 0.03M
Iodine (I): 0.03g/L
CaCl 2:?????1.85mM
pH:????????2.50-0.05
Soaking time: 40 ℃
Reaction times: 23 seconds
Wavelength: λ=590nm
Enzyme concn: 0.025AFAU/mL
Enzyme working range: 0.01-0.04AFAU/mL
If preferably other particular content then can find them and they are being incorporated herein by reference as required from the EB-SM-0259.02/01 available from Novozymes A/S.
Beta-amylase activity (DP °)
The activity of representing SPEZYME  BBA 1500 with saccharogenic power (DP) degree.It is to produce to be enough to reduce the enzyme amount that contains in the 0.1ml 5% enzyme preparation sample solution of recuding sugars of 5ml fehling's solution when under 20 ℃ sample being incubated 1 hour with the 100ml substrate.
Amylopectin enzymic activity (new amylopectin unit of enzyme Novo (NPUN)
Aureobasidium pullulans glycan substrate is measured the amylopectin enzymic activity relatively.The Aureobasidium pullulans glycan is mainly by passing through 1, the straight chain D-glucose polymer that the maltotriose glycosyl unit that 6-α-key connects forms.Inscribe-Starch debranching enzyme random hydrolysis 1,6-α-key discharges trisaccharide maltose, 6 3-α-maltotriose glycosyl-trisaccharide maltose, 6 3-α-(6 3-α-maltotriose glycosyl-maltotriose glycosyl)-trisaccharide maltose.
A new amylopectin unit of enzyme Novo (NPUN) is that inscribe-amylopectin unit of enzyme activity and contrast Novozymes A/S Promozyme D standard substance are measured.Standard conditions be under 40 ℃ and pH4.5 30 minute reaction times and with 0.7% Aureobasidium pullulans glycan as substrate.Going out amount by the red degradation of substrates product of spectrophotometry and this amount at 510nm is directly proportional with inscribe-amylopectin enzymic activity in the sample.The file (EB-SM.0420.02/01) of more specifically describing this analytical procedure is as required available from Novozymes A/S, and Denmark is included in this document herein as a reference.
Under standard conditions, a NPUN approximates to discharge has the enzyme amount of the reducing sugar that equals 2.86 micromole's glucose/minute reducing power.
The mensuration of CGTase hydrolytic activity
By as Wind etc. 1995 measure described in the Appl.Environ.Microbiol.61:1257-1265 with Paselli SA2 starch (from Avebe, The Netherlands) insulating process together in the increase of reducing power determine the hydrolytic activity of CGTase.
The dried solid of sugar cloth and dissolved is measured
Measure that sugar in the starch hydrolysate is formed and be DX with the glucose calculation of yield subsequently by HPLC.Determine the dried solid of dissolved in the starch hydrolysate (solubility) ° BRIX by measuring specific refractory power.
Material
Use following enzymic activity.Fructus Hordei Germinatus sugar α-Dian Fenmei contains the aminoacid sequence shown in the SEQ ID No:1 of WO9/943794.Glucoamylase derives from the aspergillus niger of one of aminoacid sequence shown in the SEQ ID No:2 that contains WO00/04136 or disclosed variant.Acid fungal alpha-amylase derives from black aspergillus.The bacillus α-Dian Fenmei is for containing sudden change I181 *+ G182 *The reorganization bacstearothermophilus variant of+N193F.Fungal alpha-amylase derives from aspergillus oryzae.CGTase O contains just like the sequence shown in this paper SEQ ID NO 2.CGTaseT contains the aminoacid sequence shown in disclosed and this paper SEQ ID NO 3 among (1997) Fig. 1 in Biotechnol.Lett.19:1027-1031 such as Joergensen.CGTase A contains just like the sequence shown in this paper SEQ ID NO 4.
Conventional corn starch (Cx PHARM 03406) is available from Cerestar.
Embodiment 1
Present embodiment has explained that use CGTase T and glucoamylase and acid fungal amylase change into glucose with granular starch.Join by W-Gum under agitation condition that 247.5g is commonly used that preparation has 33% dried solid (DS) granular starch slurry in the 502.5ml water.With HCl with pH regulator to 4.5.The granular starch slurry is distributed in the blue lid of the 100ml flask, and every flask contains 75g.When magnetic stirs, flask is incubated in 60 ℃ water-bath.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 1.Draw samples after 24,48,72 and 96 hours.
The used enzyme activity level of table 1. is:
????CGTase?T ????KNU/kg?DS Glucoamylase AGU/kg DS Acid fungal alpha-amylase AFAU/kg DS
????12.5 ????200 ????50
????25.0 ????200 ????50
????100.0 ????200 ????50
Use following method to measure total solids starch.By adding excessive α-Dian Fenmei (the dried solid of 300 KNU/Kg) and subsequently sample being placed the oil bath under 95 ℃ to make the starch complete hydrolysis in 45 minutes.By behind the 0.22microM membrane filtration, determine dried solid by measuring specific refractory power.
By behind the 0.22microM membrane filtration to the dried solid of the solubility in the sample determination starch hydrolysate.Determine the dried solid of solubility and measure sugar cloth by measuring specific refractory power by HPLC.The amount of glucose is calculated as DX.The result is as shown in table 2 and 3.
Table 2. dried solid of solubility under three kinds of CGTase activity levels accounts for the per-cent of total dry matter.
KNU/kgDS 24 hours 48 hours 72 hours 96 hours
12.5 68 82 ??89 ????94
25.0 76 89 ??93 ????97
100.0 83 96 ??98 ????99
The DX of table 3. solubility hydrolysate under three kinds of CGTase activity levels.
KNU/kgDS 24 hours 48 hours 72 hours 96 hours
12.5 92.6 94.5 ????95.1 ????95.3
25.0 92.4 94.8 ????95.4 ????95.5
100.0 92.7 94.9 ????95.4 ????95.4
Embodiment 2
Present embodiment has explained that use CGTase T, glucoamylase, acid fungal alpha-amylase and genus bacillus α-Dian Fenmei change into glucose with granular starch.
Be ready to contain the flask of 33%DS granular starch and preparation insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 4.
The used enzyme activity level of table 4. is:
??CGTase?T ??KNU/kg?DS Glucoamylase AGU/kg DS Acid fungal alpha-amylase AFAU/kg DS Genus bacillus α-Dian Fenmei KNU/kg DS
??5.0 ????200 ??50 ????300
Draw samples and analysis as described in example 1 above after 24,48,72 and 96 hours.The result is as shown in table 4 and 5.
The dried solid of table 5. solubility accounts for the per-cent of total dry matter.
24 hours 48 hours 72 hours 96 hours
????82.8 ????93.0 ??96.3 ??98.7
The DX of table 6. solubility hydrolysate.
24 hours 48 hours 72 hours 96 hours
????92.8 ????94.9 ????95.5 ????95.8
Embodiment 3
Present embodiment has explained that use Fructus Hordei Germinatus sugar α-Dian Fenmei, glucoamylase and acid fungal alpha-amylase change into glucose with granular starch.
Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 6.
The used enzyme activity level of table 6. is:
Fructus Hordei Germinatus sugar α-Dian Fenmei MANU/kg DS Glucoamylase AGU/kg DS Acid fungal alpha-amylase AFAU/kg DS
Flask 1 ???5000 ????200 ????50
Flask 2 ???20000 ????200 ????50
Draw samples and analysis as described in example 1 above after 24,48,72 and 96 hours.The result is as shown in table 7 and 8.
Table 7. dried solid of solubility under the activity level of two kinds of Fructus Hordei Germinatus sugar α-Dian Fenmei accounts for the per-cent of total dry matter.
MANU/kgD S 24 hours 48 hours 72 hours 96 hours
5000 20000 ????63.1 ????67.0 ????75 ????77.9 ????79.3 ????82.7 ????85.3 ????88.1
The DX of table 8. solubility hydrolysate under the activity level of two kinds of Fructus Hordei Germinatus sugar α-Dian Fenmei.
MANU/kgD S 24 hours 48 hours 72 hours 96 hours
5000 20000 ????95.2 ????93.8 ????95.4 ????94.9 ????95.3 ????94.9 ????95.5 ????94.8
Embodiment 4
Present embodiment has explained that use glucoamylase and acid fungal alpha-amylase only change into glucose with the part granular starch.
Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.The enzymic activity that in the time of 0 hour, flask is given to provide in the table 9.Draw samples after 24,48,72 and 96 hours.Analytic sample as described in example 1 above.The result is as shown in table 10 and 11.
The used enzyme activity level of table 9. is:
Glucoamylase AGU/kg DS Acid fungal alpha-amylase AFAU/kg DS
????200 ????50
The dried solid of table 10. solubility accounts for the per-cent of total dry matter.
24 hours 48 hours 72 hours 96 hours
????28.5 ??36.3 ????41.6 ??45.7
The DX of table 11. solubility hydrolysate.
24 hours 48 hours 72 hours 96 hours
????27.7 ????34.9 ????39.2 ????42.2
Embodiment 5
Present embodiment has been explained as what being measured appearred in the dried solid of solubility and DX and has been used CGTase and glucoamylase granular starch to be changed into the dependency between the 4 kinds of different CGTases (CGTase A, CGTase N, CGTase O and CGTase T) and productive rate in the process of glucose syrup.
Be ready to contain the flask of 33%DS granular starch and insulation as described in example 1 above.In the time of 0 hour, all give the CGTases of 100KNU/kg DS and the glucoamylase of 200AGU/kg DS.Draw samples is also analyzed as described in example 1 above in the time of 48 hours.To the results are shown in the table 12.
Table 12. hydrolytic activity (micromole/minute/mg protein) and dried solid of solubility (DS) and DX after 48 hours
????CGTase Hydrolytic activity Solubility DS ????DX
????CGTase?N ????0.27